Collagen and collagen matrices find many applications in surgery. They are used as a film, putty, gel and paste. Collagen (usually type 1) scaffolds are commonly used in tissue engineering, wound healing, as neurosegeneration guide substrates, and in reconstructive surgery and dermatology. However, most available preparations have poor biomechanical properties compared to tissues they are intended to replace. For that reason, collagen constructs are subject to laboratory manipulations. The strength of collagen fibrils which under natural conditions is not great, can be increased by covalent crosslinks within and between the constituent collagen molecules. Collagen can self-assemble through an enzymatic formation of intermolecular cross-links leading to a head to tail arrangement within the fiber. Gluteraldehyde is one of the most common reagents for crosslinking collagen from collagen gels.
Collagen gel is a convenient, abundant and commonly used biomatrix in laboratory studies. Collagen used in the laboratory studies is obtainable commercially in large quantities. It is extracted from animal sources by acid hydrolysis, producing acid soluble collagen which has to be neutralized before use.
The mechanical properties of collagen gels have been extensively investigated. In this connection, anisotropic hyperelastic constitutive models are used. These include collagen fiber dispersion. The response of the model captures both an isotropic and anisotropic component. The latter assumes collagen fibers to be active only in extension.
Numerous studies with collagen gels and other purified collagen substances were directed at producing collagen scaffolding. These efforts were largely unsuccessful, primarily because of poor biomechanical characteristics of constructed scaffoldings, compared to structures they were intended to replace.
Clearly something else had to be tried. Not all collagen-containing membranous structures are architecturally alike. Skin dermis is made up of two layers with elastic fibers interspersed among dense collagen fibers. Fascia is made up of parallel collagen columns held together by loose fibrous tissue. Like dermis, fascia lata is composed of two layers. Tendons have relatively few cells and an abundant collagenous material. Dura mater is a specialized structure with collagen fibers oriented in lamina. The architecture of collagen fibers of dura mater is unique compared to other membranous structures. Dura mater collagen presents an anisotropic structure.
Due to the variety of different collagen types and structures and the use of a variety of sources with degrading and harsh methods of cleaning and processing the material, the results with collagen gels and other purified collagen based material needs improvement due to the generally poor results with collagen scaffolding.
One such improvement is in the use of strong magnetic fields.
Magnetic fields and their influence on mammalian cells have been studied over several decades by a number of investigators. There have been numerous attempts to demonstrate significant effects on biologic systems. Magnetic fields were reported to effect cellular metabolism. Unequivocal effect on intact mammalian cells by magnetic fields was demonstrated by Malinin et al in 1976. These investigations showed that strong magnetic fields (5000 oersted) can induce morphologic and physiologic transformations of mammalian cells. They concluded that the transformations were due to magnetic field effects alone. This is important, as subsequently Brighton postulated that the effects of pulsed electromagnetic fields are due to the electric field, and not the magnetic field. The difference between the magnetic and the electric field is, that in a magnetic field force on a charged particle is due to charged particle movement while the force imparted by an electric field on a charged particle is not due to the charged particle's movement. From a practical standpoint, the difference is important. Magnetic fields at commonly employed frequencies penetrate biological systems, including the human body to any depth; electrical fields do not, necessitating placement of electrodes near the object to be effected. In living cells, static magnetic fields decrease cell death by apoptosis. Magnetic control of osteoblast collagen orientation has also been reported.
In non-viable systems of collagen gel, orthogonal scaffold of magnetically aligned collagen had been attempted to provide for substitution of corneal stroma. Strain induced alignment of collagen gels had been reported. Static magnetic field effects on cells of pheochromacytoma cell line demonstrated effect on adenosine receptors. Byphasic nature of collagen gels is used to explain the mechanical behavior of collagen scaffoldings under mechanical stress. Static magnetic fields are reported to have little effect on cells. All of the above cited studies were performed either on living mammalian cells or on collagen gels with collagen of bovine origin subjected to acid hydrolysis or other modes of harsh treatment such as heat.
In the prior art, thermally assisted pulse electromagnetic field stimulation is disclosed in U.S. Pat. No. 8,460,167. The effect on the joint treated by the method described in the embodiments is produced by electric rather than magnetic fields. In a Japanese patent JP2006-280222 entitled, “Magnetic Circuit For Arranging Molecules Such As Collagen And Cell And Apparatus Using The Same”, the inventors disclose a magnetic circuit characterized by using a permanent magnet having a generated magnetic field strength of >=2T as a magnetic field-generating source to arrange collagen, cells and the like, and an apparatus using the magnetic circuit. The collagen subjected to the disclosure in this invention was that of osteoblast in cell culture. The magnetic field was of high intensity 2.0 T and 2.5 T derived from permanent magnets. The effect was observed only on mouse osteoblast cells secreting collagen with respect to their orientation. Collagen used was of animal origin in the form of a gel. The disclosures are strikingly different from the embodiments of the present invention. The Hajme invention is based on viable cells and bovine gel collagen.
In WO2013069661, entitled “Vascular Progenitor Cell Sheet Derived From Induced Pluripotent Stem Cells, And Production Method Therefor”, the invention addresses the problem of providing a vascular progenitor cell sheet derived from induced pluripotent stem cells, which has the strength to tolerate practical applications and exhibits a high treatment effect. This vascular progenitor cell sheet derived from induced pluripotent stem cells is prepared by performing: (1) a step for preparing magnetically labeled Flk-1 positive cells derived from induced pluripotent stem cells; (2) a step for preparing a mixture of the Flk-1 positive cells and a gel material including type I collagen, laminin, type IV collagen and entactin as active ingredients, and then disseminating the mixture in a culture vessel; (3) a step for drawing the Flk-1 positive cells in the mixture to the culture surface of the culture vessel by application of a magnetic force to form a multi-layered cell layer; and (4) a step for gelling the gel material. The WO2013069661 invention deals with magnetically labeled cells. The method aids in cell separation of animal origin collagen type I and IV used for cell propagation.
In US2010074874, “Synthetic Multi-Layer Structures Comprising Biopolymer Fibres”, the invention relates to a method for the preparation of a synthetic multi-layer structure comprising biopolymer fibers, wherein the biopolymer fibers in each layer are unidirectionally and uniformly oriented, which comprises successive polymerization of layers of a biopolymer fiber forming solution in the presence of a magnetic field, wherein the fiber orientation in at least one layer differs from that in at least one of its superior and/or inferior layer according to an angle alpha. The invention also relates to a biological tissue-like multi-layer structure comprising the synthetic multi-layer structure and cells inoculated therein, such as an orthogonal multi-layer collagen and/or fibrin tissue-like cornea, and the method for its preparation. The invention deals with synthetic biopolymer structures.
U.S. Pat. No. 6,194,182, “Magnetically oriented tissue-equivalent and biopolymer tubes”, discloses tissue-equivalent and biopolymer tubes which are oriented by a magnetic field. These oriented fibrils provide enhanced mechanical properties to the tissue-equivalent and biopolymer tubes. One such tissue-equivalent tube includes a body of collagen with mammalian tissue cells interspersed therein. The collagen fibrils are circumferentially oriented within the tubular body by a magnetic field, thereby inducing circumferential orientation of the cells. Methods of making magnetically oriented tissue-equivalent tubes and biopolymer tubes are also disclosed. The invention deals primarily with biopolymer tubes loaded with cells and collagen fibrils, not fibers from collagen gels. The type of magnetic field used to orient these is not disclosed. The invention described deals with biopolymers, viable cells and subcellular components of collagen gels of animal origin.
The present invention has the objective of solving these issues of poor collagen scaffolding by a clearly unique combination using human collagen harvested and processed to maximize structural scaffolding.